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Engineered CRISPR-Cas9 Variants Are Activated by Proteases

NEW YORK (GenomeWeb) – Investigators from the University of California, Berkeley and the Lawrence Berkeley National Laboratory have engineered CRISPR-Cas9 variants that are inactive until they encounter viral proteases.

As the researchers wrote today in Cell, a circular permutation approach allowed them to rearrange Cas9 into "an advanced platform for RNA-guided genome modification and protection." Further, they noted that circular permutation enabled the development of protease-sensing Cas9s (ProCas9s), a class of single-molecule effectors with programmable inputs and outputs that can orchestrate a cellular response to pathogen-associated protease activity.

Developing an optimized Cas9 architecture for controlled nuclease activity and facilitating efficient construction of fusion proteins would expand and improve future applications, the researchers noted. One way of doing this is by protein circular permutation (CP) — "the topological rearrangement of a protein's primary sequence, connecting its N- and C-terminus with a peptide linker, while concurrently splitting its sequence at a different position to create new, adjacent N and C termini," they wrote. This type of repositioning changes the way a protein behaves.

For this study, the researchers used circular permutation to re-engineer the molecular sequence of Cas9 in order to better control its activity and create a more optimal DNA-binding scaffold for fusion proteins. They created libraries and combined them with high-throughput fitness assays and deep sequencing to define a set of possible Cas9 circular permutants, collectively called Cas9-CPs.

Interestingly, in one assay, the researchers observed "surprisingly high" genome editing efficiency in the Cas9-CPs compared to wild-type Cas9. And while they saw more variation in other experiments, four of the CP variants they tested showed 80 percent or more of wild-type Cas9 activity. This demonstrated that Cas9 could be circularly permuted to create novel proteins that may maintain wild-type like levels of DNA binding and cleavage activity.

"Our analysis shows that Cas9 is highly malleable to circular permutation, and several regions of the protein … possess hotspots that can be opened at numerous positions to generate a diversity of Cas9-CPs," the authors wrote. "We further show that engineering of the linker sequence with site-specific protease sequences, derived from a variety of pathogenic plant and human viruses, yields 'caged' pro-enzyme Cas9 variants that can be activated by proteolytic cleavage."

The resulting "uncaged" enzymes, which the team called ProCas9s, were capable of responding to the presence of numerous, distinct families of viral proteases in E. coli and various mammalian cell types, the researchers found. Through various experiments, they also determined that ProCas9s can be stably integrated into mammalian genomes to sense, record, and respond to endogenous or exogenous protease activity.

"A molecular sensor, such as ProCas9, could actuate many types of outputs. One unique effect would be to induce cell death upon sensing viral infection, as a form of altruistic defense," the authors wrote. "The system must remain off to minimize genomic damage, yet be vigilant to respond to a stimulus."

Indeed, when they tested whether such a system could be introduced into the genome and programmed in such as a way as to respond only when needed, the researchers found that it could be stably integrated into the host genome to detect predefined viral protease activity and only activate itself in order to defend the host against the viral invader.

"The ProCas9s' ability to serve as a detector of pathogen activity is intriguing as it could enable their use as a fully modular, genomically encoded immune system with both a designable input and programmable output," the authors concluded. "We have demonstrated an initial proof-of-concept for a fully synthetic and customizable resistance gene. Hence, it should be straightforward to transition this self-targeting system into a platform that can induce expression or suppression of genes to mount a systemic immune response, or to activate a synthetic cellular program to track pathogen invasion. Such a strategy for pathogen detection is broadly applicable, as many pathogens express proteases during host infection."

Further, they noted, the Cas9-CPs serve as a diverse set of protein scaffolds for advanced CRISPR-Cas fusion proteins.